The present invention relates to methods and apparatus for separating analytes by capillary electrophoresis. In particular, the present invention relates to capillary array electrophoresis and a method of performing high-speed, high-resolution separations of DNA.
The Human Genome Project is driving the development of instrumentation, reagents and methods to achieve the challenging goal of sequencing 3 billion base pairs of human DNA. One new and effective separation technique for nucleic acid analysis is capillary electrophoresis. The use of this technique for DNA sequencing was first demonstrated in 1990 with the separation of approximately 350 bases on cross-linked gels in just over an hour. Since then, a number of groups have improved this technique. Early improvements, for example, utilized in situ polymerized linear polyacrylamide gels and were successful in obtaining high resolution separations. Polymerization reproducibility was problematic with such methods, however, which has driven the development of replaceable gel matrices. One can now read sequences of approximately 1000 bases in 60-90 minutes using capillaries filled with replaceable linear polyacrylamide solutions.
Adapting capillary electrophoresis to high throughput applications requires that one perform separations on arrays of capillaries. The technique, called capillary array electrophoresis, may be performed in conjunction with confocal fluorescence scanning detection, as described in U.S. Pat. No. 5,091,652, the entirety of which is incorporated herein by reference. An improved, four-color version of such detection is described in U.S. Pat. No. 5,274,240, the entirety of which is also incorporated herein by reference.
In the development of capillary array electrophoresis systems, the goal has typically been to run and detect 96 capillaries in parallel. One such system has been commercially developed; it provides automated sample and gel loading of 96 capillaries and is used by high throughput genome centers and pharmaceutical companies. While such systems have proven valuable, it is still desirable to increase their processing power. While these systems may be able to read over 1000 bases per hour, this figure is not so impressive when one considers that the human genome comprises over 3 billion bases.
It is an object of the present invention to provide an apparatus capable of performing capillary array electrophoresis within more than 96 capillaries, and to detect the analytes thus separated. It is a further object of the invention to perform such separation at high speed, suitable for the most demanding high-throughput applications. It is a particular object of the invention to enable detection of over 1,000 bases per hour, and to provide a system compatible with known automation techniques. Yet another object of the invention is to provide for an improved detection apparatus, capable of detecting in four colors the analytes separated.
Described herein is an improved rotary confocal fluorescence scanner capable of detecting analytes separated on over 1,000 capillaries simultaneously. This system uses a confocal microscope objective and mirror assembly that rotates inside a vertical ring of capillaries to provide rapid and efficient excitation and detection of fluorescently labeled fragments separated within a cylindrical capillary array. Use of automated procedures to load and run all capillaries permits one to read more than 350,000 base pairs of raw sequence data per hour.